Carlos A. Nagle

Effects of melatonin on neurotransmitter uptake and release by synaptosome-rich homogenates of the rat hypothalamus.

Preincubation of synaptosome-rich homogenates of rat hypothalamus with melatonin resulted in significant decreases of norepinephrine, serotonin, dopamine and glutamate uptake. Melatonin inhibition was noncompetitive; apparent Kms of initial uptake processes were: (2.5 +/- 0.3) x 10(-7) M for norepinephrine, (2.6 +/- 0.3) x 10(-7) M for serotonin, (2.4 +/- 0.4) x 10(-7) M for dopamine and (1.0 +/- 0.3) x 10(-7) M for glutamate. Apparent Kis for melatonin inhibition of transmitter uptake were: 0.64 +/- 0.14 mM (norepinephrine), 0.23 +/- mM (serotonin), 0.51 +/- 0.08 mM (dopamine) and 1.21 +/- 0.10 mM (glutamate). Transmitter release evoked by increasing [K+] in medium to 30 mM was augmented by melatonin in a dose-dependent manner. Maximal effects were observed on serotonin release. Accumulation of 3H-melatonin within synaptosome-rich homogenates did not exhibit differences between 0 and 37 degrees C, indicating that the uptake of the hormone was not an active process. These results suggest that exogenously-administered melatonin may affect neurotransmitter accumulation and release in the hypothalamus by modification of the transmitter uptake mechanism rather than by competition with the transmitter for its uptake pump.

Control of estrogen and androgen receptors in the rat pineal gland by catecholamine transmitter

Abstract Sucrose gradient studies of rat pineal cytosol incubated with 3 H-estradiol (female pineals) or 3 H-5 α -dihydrotestosterone (male pineals) revealed a radioactivity peak in the 8 S region which disappeared after superior cervical ganglionectomy or incubation with excess unlabeled hormone. Ganglionectomy decreased significantly estradiol and testosterone uptake by the pineal gland in vitro as well as high affinity binding to pineal cytoplasmic and nuclear components. Norepinephrine treatment counteracted all the effects of ganglionectomy but was unable to modify hormone uptake and binding by the pineal gland of sham-operated controls. Pre-treatment with actinomycin D or propranolol but not with phentolamine impaired norepinephrine effects; propranolol blockage however was only partial. Administration of isoproterenol, L-dopa or phentolamine increased hormone uptake by denervated pineals. The effects of isoproterenol were also observed in vitro and were blocked by propranolol. These results indicate that sex steroid receptors in the pinealocytes are controlled by norepinephrine via beta-adrenergic receptors and that depletion of neural norepinephrine enhanced responsiveness of pineal hormone receptors to exogenous catecholamines.

Effects of estradiol on melatonin and protein synthesis in the rat pineal organ.

The effects of estradiol on pineal function haveinsofar been studied by administering supramaximal, non-physiological doses of the hormone. The present experiments were undertaken to assess estradiol-

Gonadal steroids as modulators of the function of the pineal gland

Sex steroids affect pineal function. Castration or the administration of estradiol, testosterone, or progesterone bring about changes in pineal melatonin synthesis, as well as in other aspects of pineal activity in rats. This paper deals with some of the mechanisms underlying steroid action on pineal metabolism. Estradiol is avidly taken up in vivo and in vitro by the pineal gland. A cytosol binding protein having a Kd of, (0.27 ± 0.08) × 10−9M for estradiol and requiring SH groups for full expression of binding activity was detected in pineal homogenates. Estradiol uptake by the pineal and the uterus of mature rats attained their minima on the day of proestrus. A priming dose of estradiol increased high affinity binding sites for estradiol in pineal and uterine cytosol by about 150%. Testosterone is also readily taken up and retained in vivo and in vitro by the pineal of orchiectomized rats. Saturation kinetics analysis of androgen binding to bovine pineal cytosol indicated a Kd of 1.57 × 10−9M for testosterone and 0.36 × 10−9M for 5α-dihydrotestosterone. The cytoplasmic binder was shown to be a protein and to require SH groups for binding activity. Testosterone was metabolized by the pinealocytes into 5α-dihydrotestosterone, 5α-androstanediol, androstenedione and 5α-androstanedione; 5α-dihydrotestosterone was the major metabolite in the nuclei of pineal cells. Superior cervical ganglionectomy or exposure of rats to light increased androstenedione synthesis, whereas ganglionectomy or exposure to darkness decrease 5α-androstanedione synthesis. Progesterone is taken up by the pinealocytes in vitro up to a tissue concentration 18-fold that of the media. [3H]Progesterone is converted in the pinealocytes into 5α-pregnanedione and 3α-hydroxy-5α-pregnan-20-one. These data suggest that the early steps of sex steroid action on pineal cells resemble those of steroid target tissues.

Effects of Castration and Testosterone Administration on Pineal and Retinal Hydroxyindole-O-Methyl Transferases of Male Rats

The effects of castration andtestosterone treatment on pineal and retinal hydroxyindole-O-methyl transferases (HIOMT) were investigated in male rats. Pineal and retinal HIOMT activities decreased 24–7

Testosterone effects on protein synthesis in the rat pineal gland. Modulation by the sympathetic nervous system

Abstract The administration of 0.5 mg of testosterone propionate (TP) for 3 days to castrated male rats caused 3H-leucine incorporation into pineal proteins to increase significantly by 79%. TP effects depended on time of administration; rats receiving TP at 06.00 h exhibited a significant 150% increase in pineal protein synthesis 24 h later whereas rats injected at 14.00 h only showed a 54% increase in 3H-leucine incorporation into proteins. Superior cervical ganglionectomy decreased pineal testosterone uptake in vitro by 21% and pineal protein synthesis by 27%; in addition it blocked the stimulatory effects of TP on protein synthesis. Ganglionectomy also modified the in vitro metabolism of testosterone by pineal cells; it increased the amounts of 3H-androstenedione and decreased 3H-5∝-androstanedione extracted from pineal glands incubated with 3H-testosterone. These results indicate that the sympathetic nervous input reaching the pineal via the superior cervical ganglia is important to modulate the early steps of androgen action on the pinealocytes.

Mitochondrial Divergence Between 2 Populations of the Hooded Capuchin, Cebus (Sapajus) cay (Platyrrhini, Primates)

We analyzed the molecular divergence of 2 separate populations of Cebus apella paraguayanus, recently considered a junior synonym of Cebus cay, and estimated its time of separation from C. apella. Cytochrome b DNA from 23 C. cay from Brazil and 9 from Paraguay showed 24 haplotypes (20 and 4, respectively), accounting for 29 variable sites (19 transitions and 10 transversions), with 40.0%, 26.7%, and 33.0% replacements at first, second, and third codon positions, respectively. Genetic distance between haplotypes averaged 0.5%, with 1.1% between C. cay populations. Phylogenetic reconstructions and median joining separated C. cay from Brazil and Paraguay. Neighbor joining showed C. cay and C. apella as sister groups, although C. cay and C. apella collapsed in maximum parsimony and maximum likelihood topologies. Analysis of molecular variance showed the highest variance component between C. cay populations, and mismatch distribution indicated that this species suffered a recent demographic expansion. Divergence time estimates suggested that the 2 populations of C. cay split in the Pleistocene, a period of repeated glaciation events leading to drastic changes in the vegetation composition of different biomes.

Rat Brain Norepinephrine Release During Progesterone-Induced LH Secretion

The administration of 1.5 mg of progesterone to ovariectomized estrogen-primed rats induced a surge in plasma norepinephrine (NE) preceding the pituitary LH release. The injection of reserpine (2 mg/kg) 2 h prior to progesterone completely blocked the progesterone effects on both NE and LH plasma surges. On the other hand, the adminitration of phenoxybenzamine prior to progesterone, blocked the steroid effect on LH without inhibiting the plasma NE rise. Contrarily, the alpha-blocker was able to induce a rapid increase in the amine levels 2 h after its injection. The determination of NE in the anterior hypothalamus of these rats revealed that simultaneously with the plasma NE rise induced by progesterone there was a fall in the hypothalamic amine levels. Both reserpine and phenoxybenzamine caused a depletion in the amine content of the anterior hypothalamus. In experiments measuring the arteriovenous difference in the concentration of NE across the brain following progesterone-induced LH release, it was found that most NE comes from the brain. The amine rise was evident in the jugular vein while the arterial NE concentration showed a slight increase. These findings raise the possibility that the changes in circulating NE may reflect an enhanced noradrenergic activity occurring in the brain from which the LH surge results.

Uptake and effect of 17β-estradiol on pineal hydroxy-indole-o-methyl transferase (HIOMT) activity

In the present work we have studied a) the uptake of estradiol by the pineal gland and b) the effects of exogenous estradiol and norepinephrine on pineal hydroxy-indole-O-methyl transferase (HIOMT) activity. The uptake of estradiol by the pineal gland, the uterus and the cerebral cortex of castrated rats was determined 15–240 min after the intraperitoneal administration of a single dose of [6,7−3H] 17β-estradiol. The pineal took up and retained significantly more [3H] estradiol per mg of tissue than either the uterus or the cerebral cortex (p<0.01). The simultaneous injection of norepinephrine plus labeled estradiol did not modify significantly the estradiol uptake by the pineal gland. Estradiol administration for 3 days decreased pineal HIOMT activity (p<0.01), this effect being reversed by the simultaneous injection of norepinephrine. When norepinephrine was injected alone, no effects on pineal HIOMT were observed. These results suggest the existence of an estradiol receptor system in the rat pineal gland.

Uptake of estradiol by the rat pineal organ. Effects of cervical sympathectomy, stage of the estrous cycle and estradiol treatment

Abstract Rat pineal organs of spayed rats took up and retained estradiol in vitro up to 32-fold the concentration present in the incubation media. This phenomenon was maximum at 37°C and after 2-h incubations. Most (86–91%) of [ 3 H] radioactivity recovered from the incubated pineals was identified as estradiol by thin-layer chromatography. Treatment with dextran-coated charcoal of nuclei-free pineal homogenates incubated with [ 3 H] estradiol of different SA uncovered a high affinity, low capacity binding of estradiol to cytosol components. Uptake of estradiol by the nuclear fraction also proceeded in a saturable fashion. Similar findings were made in uterine homogenates of spayed rats. Estradiol uptake by the pineal organ and the uterus of cycling rats varied as a function of the stage of the estrous cycle, maxima being observed in diestrus and minima in proestrus. The administration of a priming dose of estradiol benzoate to spayed rats caused high affinity binding components of the pineal cytosol to increase by about 150%. Nuclear binding of estradiol was also increased by the estradiol priming dose. Pineal denervation, i.e., by superior cervical ganglionectomy, caused pineal estradiol uptake to decrease significantly by about 20%. These data suggest that the early steps of estradiol action on the pineal organ may resemble those of the uterus.